PROJECT SUMMARY Reflexive motor behaviors of the intestine including peristalsis are controlled by the enteric nervous system (ENS); a complex neural network embedded in the gut wall. Perturbations within the ENS contribute to the development of dysmotility in irritable bowel syndrome, inflammatory bowel disease, and severe motility disorders such as chronic intestinal pseudo-obstruction, but the mechanisms responsible for persistent changes in enteric neural circuitry are unknown. Recent data show that enteric glia, non-neuronal cells that surround enteric neurons, regulate neuronal excitability and contribute to neuroinflammation. The overall goal of this proposal is to define how specialized interactions between enteric glia and neurons regulate motility and how alterations in those mechanisms contribute to disease. This proposal tests the central hypothesis that enteric glia are specialized to potentiate the activity of ascending excitatory neural pathways involved in normal contractile motility, and that disruption of this regulatory system by inflammation contributes to neuronal hyperexcitability. This dual hypothesis will be tested in two specific aims that utilize genetically encoded calcium indicators to study neuron-glia interactions, glial chemogenetic actuators to study how glia modulate specific types of enteric neurons, and a post-inflammatory model of enteric neuroplasticity to study how glia contribute to neuronal hyperexcitability following inflammation. Aim 1 will test the hypothesis that enteric glia are specialized to sense excitatory neurons and potentiate ascending neural pathways involved in the contractile phase of motility. Aim 1.1 will use genetically encoded calcium indicators to study glial recruitment by polarized neural pathways in motility reflexes. Aim 1.2 will combine the chemogenetic activation of enteric glia with neuronal and glial imaging using genetically encoded calcium indicators to test the hypothesis that glia differentially affect subsets of enteric neurons. Aim 2 will test the hypothesis that glia contribute to neuronal hyperexcitability following colitis by increasing positive feedback to excitatory neurons and by reducing inhibitory feedback from inhibitory neurons. Aim 2.1 will study how altered interactions between glia and excitatory neurons contribute to neuronal hyperexcitability following colitis. Aim 2.2 will use mutant mice and selective drugs to study how glia contribute to neuronal hyperexcitability through interactions with inhibitory neurons. The results of this study will provide novel insight into glial mechanisms that regulate the excitability of enteric neural circuits. A better understanding of the glial mechanisms that regulate motility will facilitate the development of therapeutics for dysmotility by revealing novel targets to modify gastrointestinal reflexes.